NIV Congres

Background Part of the antigen-specific memory T-cells generated against pathogens, such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can also respond to allo-antigens, so called heterologous immunity. In kidney transplant recipients, the presence of virus specific T-cells with cross-reactivity to allo-antigens of donor origin might impair allograft function and survival. To study the role of heterologous immunity we developed a method to analyze the frequency and function of virus-specific T-cells with cross-reactivity against allo-antigens.

Methods We used peripheral blood mononuclear cells (PBMCs) from 5 healthy individuals known to have circulating CMV- or EBV-specific T-cells, which could be detected by tetramer-staining. PBMCs were used directly, or were labelled with CFSE and cultured for 6 days either with allogeneic stimulator cells in a mixed lymphocyte reaction (MLR) or with viral peptide and IL-2 to expand respectively allo- and virus-specific T-cells. Cells were (re-) stimulated for 6h with allogeneic cells or peptide loaded-autologous PBMCs and analyzed by flow cytometry. We identified cross-reactive CD8+ T-cells as tetramer+ and CFSEdim in MLRs. 

Results Viral-antigen dose-dependently induced cytokine production and expression of the degranulation marker CD107α in virus-specific T-cells analyzed directly or expanded by either viral- or allo-antigen stimulation. Interestingly, EBV-specific T-cells, which have an effector-memory phenotype, produced both IFNγ and IL-2, whereas effector type CMV-specific T-cells produced only IFNγ. Re-stimulation with allo-antigen induced IFNγ in a small but specific percentage of T-cells which had expanded upon stimulation by viral peptide. These findings confirm that cross-reactive T cells responding to both allo- and viral- antigens can be detected in vitro by proliferation, IFNγ production and degranulation. We could not identify cross-reactive allo-specific T-cells by intracellular cytokine staining directly ex vivo, suggesting that the activation state of cells determines their capacity to secrete cytokines.   

Conclusion Together these results show that frequency and function of cross-reactive T-cells can be analyzed in vitro.